Neuroglobin Plays a Protective Role in Arsenite-Induced Cytotoxicity by Inhibition of Cdc42 and Rac1GTPases in Rat Cerebellar Granule Neurons

Abstract

Background and Aims: We have previously shown that neuroglobin (Ngb) expression can be regulated by sodium arsenite (NaAsO₂) exposure in rat cerebellar granule neurons (CGNs). However, the precise molecular mechanisms of Ngb action are largely unknown. Ras homolog (Rho) guanosine triphosphatases (Rho GTPases) are involved in the regulation of a number of cellular processes, including cell cytotoxicity. It has been reported that Ngb can act as a guanine nucleotide dissociation inhibitior (GDI) role to inactivate Rho GTPases. Therefore, we investigated Rho GTPases activation induced by NaAsO₂ exposure in rat CGNs and effects of Rho GTPases activation on the cells. We also investigated the role of Ngb in this process. Methods: Primary cultures of CGNs were prepared from 7-day-old Wistar rat pups. The cytotoxic effects of NaAsO₂ on CGNs were evaluated using the Cell Counting Kit-8 assay and TUNEL staining. RNA interference technology was used to silence Ngb, and the subsequent effects were evaluated by quantitative RT-PCR and Western blot. Cdc42 and Rac1 activation were measured by pull-down assay and Western blot. Results: NaAsO₂ induced cytotoxicity in rat CGNs, increased GTP-bound form of Cdc42 and Rac1 GTPases in the cells. Furthermore, inhibition of Cdc42 or Rac1 activity using the inhibitor ZCL278 or NSC23766 decreased apoptosis and increased cell viability in the cells exposed to NaAsO₂. Using siRNA-mediated knockdown, we show that NaAsO₂-induced cytotoxicity was exacerbated, activation of Cdc42 (GTP-Cdc42) and Rac1 (GTP-Rac1) was increased in Ngb RNA silencing cells. Conclusions: cytotoxic effects of NaAsO₂ on rat CGNs is induced at least partly by Cdc42 and Rac1 activation, and Ngb can inhibit Cdc42 and Rac1 activation to play protective role in rat CGNs exposed to NaAsO₂.

Neuroglobin, a Factor Playing for Nerve Cell Survival

“…These results are in line with previous data showing that, in cultured cells, overexpression of NGB protected against hydrogen peroxide [35]. Cerebellar granule neurons were recently used [36,37] to study NGB in relation to neurotoxic challenges induced by sodium arsenite (NaAsO 2 ). RNA interference technology was used to silence NGB and the results indicated that in silenced cells the NaAsO 2 -induced cytotoxicity was exacerbated, suggesting a protective role for NGB in this pathological condition.…”

“…NGB treatment was able to stabilize mitochondrial membrane potential [34] and NGB overexpression was associated with reduced mitochondrial DNA damage [50]. Furthermore, as a consequence of NGB overexpression, most of the studies reported a significant decrease of death signaling and a downregulation of molecules involved in the apoptotic cascade [32,36,37,42,44,45,50]. …”

Drugging the Small GTPase Pathways in Cancer Treatment: Promises and Challenges

“…For example, ZCL278 can restrain the migratory and invasive characteristics of the prostate cancer cell line PC3 in vitro [139]. In addition, it can also prevent the toxicity of sodium arsenite (NaAsO 2 ) on astrocytes and cerebellar granule neurons, encouraging its use to alleviate arsenic poisoning on the nervous system [141,142].…”

Upregulation of Neuroglobin Promotes Tm3 Leydig Cell Viability

“…Additionally, low levels of TUNEL positive cells in NGB transduced cells demonstrated the anti-apoptotic role of NGB in Leydig cells. Knockdown of NGB in neurons increased TUNEL positive cells in arsenite induce toxicity indicating the protective role of NGB against apoptosis (Liu et al 2015). Anti-apoptotic activity was confirmed by gene expression analysis of pro-and antiapoptotic genes including BAX, p53, caspase-3 and Bcl-2.…”